Colony specificity and starvation-driven changes in activity patterns of the red ant Myrmica rubra

Although the activity levels of insect societies are assumed to contribute to their ergonomic efficiency, most studies of the temporal organization of ant colony activity have focused on only a few species. Little is known about the variation in activity patterns across colonies and species, and in different environmental contexts. In this study, the activity patterns of colonies of the red ant Myrmica rubra were characterized over 15 consecutive days. The main goals were to evaluate the colony specificity of the activity patterns and the impact of food deprivation on these patterns. We found that the average activity level varied across colonies and remained consistent over 1 week, providing evidence that the activity level is a colony-specific life trait. Furthermore, all colonies applied an energy-saving strategy, decreasing their average levels of activity inside the nest, when starved. Starvation induced no consistent change in the activity level outside of the nest. An analysis of activity time series revealed activity bursts, with nestmates being active (or inactive) together, the amplitudes of which reflected the ants’ degree of synchronization. Food deprivation increased the amplitude and number of these activity bursts. Finally, wavelet analyses of daily activity patterns revealed no evidence of any periodicity of activity bouts occurring inside or outside of the nest. This study showed that M. rubra ant colonies are characterized by specific activity levels that decrease in response to starvation with the adoption of an energy-saving strategy. In addition, our results help to understand the functional value associated with synchronized and/or periodic fluctuation in activity, which has been debated for years.


Introduction
The remarkable ecological success of social insects depends on their high efficiency in performing collective tasks, such as the exploration of new areas, the exploitation and sharing of resources, the building of a common nest, and the defense of a common territory. As a correlate, popular beliefs have often acknowledged insect societies as the paradigm of industrious animals being extremely active throughout their life. This has led to some misconception about the actual activity level of insect societies and the distribution of activity among colony members. Several studies have provided striking examples of inactivity, with up to 50 With this study, we aimed to fill this knowledge gap by characterizing the activity patterns of the red ant species Myrmica rubra, which deeply differs in its behavioral ecology, its pace of life as well as its colony size, while showing a high diversity of nestmate relatedness across colonies [48][49][50]. We recorded colony activity over several successive days to clarify the stability and colony specificity of the global activity level. We also investigated the flexibility and resilience of activity levels with exposure to fluctuations in resource availability, nutritional needs, and in-nest tasks to be performed (e.g., food sharing). To this aim we analyzed activity patterns of ant colonies under a stable condition of satiation, a stressful condition of multiple-day food shortage, and a condition of recovered satiation through the exploitation of a newly available food source. All the activity patterns measured inside the nest will be characterized in terms of their average level, periodicity and synchronization of activity bouts. We examined whether a greater need for nutrients in starved colonies led to an increase in foraging activity to find new resources and/or a decrease in activity inside the nest to preserve energy. Specifically, we examined whether a decrease in the average in-nest activity level was compensated by greater synchronization of activity bouts among nestmates to facilitate task achievement [20,46]. For the recovery of satiation following the discovery of a new food source, we quantified whether an increase in food-sharing tasks was accompanied by an increase in the average in-nest activity level and/or a larger number of activity peaks. The study data provide a general overview of variation in ant colony activity patterns inside and outside of the nest over time, spontaneously and in response to changing food demands and resource availability.

Method
The studied species is the red ant, Myrmica rubra (Linnaeus, 1758) (Hymenoptera: Formicidae). Ant colonies were collected from the year 2018 to 2021, during the summer, in woodlands located at Sambreville (Namur district: N 50˚25.210'; E 004˚37.878') and Aiseau-Presles (Hainaut district: N 50˚25.657'; E 004˚35.764') in Belgium. These field colonies were kept under controlled laboratory conditions (21˚C ± 1˚C; 50% ± 5% humidity and 12L-12D daynight cycle) for a minimum duration of 30 days before carrying out the experiments. Each colony was used no longer than 10 months after its collection in nature. We provided colonies with water, sucrose solution (0.3M) and freshly killed mealworms (Tenebrio molitor) ad libitum. Thereafter, these field colonies were divided into standardized experimental colonies that consisted in 200 workers, one queen and 20 larvae (from the 1 st to the 3 rd larval instar). As for the 200 workers, we aimed to standardize the ratio of inner workers and foragers in the experimental colonies by always taking 140 individuals from the nesting tubes of field colonies and 60 individuals from their foraging area.
Each experiment took place in a 22x38cm plastic tray (Fig 1) with border walls covered with Fluon1 (polytetrafluoroethylene) to prevent ants from climbing out. The experimental nests were made from 2mm thick, laser-cut Plexiglas squares that provided the ants with a nesting area of 30.25cm 2 (5.5x5.5cm). The single entrance to the nest was a 1.0cm long and 0.5cm wide opening. The nest ceiling consisted in another Plexiglas square and was covered by a red filter paper to create darkness inside the nest. The nest floor was made of plaster and humidified from below with a cotton wool wick that was connected to a water reservoir. The nests were off centered in the tray and placed 2.0cm away from its back side with the nest entrance facing the remaining space of the tray over which the ants could forage. The nest area was chosen in order to host up to 200 ants without having ants climbing on each other under the nest ceiling while still leaving some space for workers to move inside the nest.

Experimental procedure
An experiment lasted for 15 days on each tested ant colony. Before the experiment, the ants were placed in the experimental tray, and we let them settle inside the Plexiglas nest for 24 hours before any data collection. For the next 15 days, on each day, we video-recorded the nest interior as well as the foraging area for 2 minutes, every 10 minutes. The experimental trays were illuminated during the whole experiment by a LED lighting in order to get a recording of ant activity that covers a full 24h-period. The experimental colonies were given mealworms, sucrose solution (0.3M) and water ad libitum for the first 4 days (Fig 2). This baseline period allowed us to quantify and analyze the activity patterns of ant colonies under stable conditions of satiation (P1). During these days of colony satiation, new mealworms were provided each day at 10.30am to make sure that the quality of the food stayed satisfactory and similar over time. At 10am on the 5 th day of the experiment, we removed the sucrose solution and the mealworms from the foraging area for seven consecutive days. This starvation period (P2) allowed us to detect possible changes in the colony activity due to food deprivation. Both mealworms and sucrose solution were given back to Each tray contains the red sheet-covered experimental nest, a sugar tube, a water tube and a Petri dish with mealworms. A camera is set over the nest and another one over the foraging area.
https://doi.org/10.1371/journal.pone.0273087.g001 the colony at 10am on the 12 th day of the experiment with the ants having access to food ad libitum until the 15 th day. This recovery period of satiation (P3) lasted three days and allowed to capture the impact of food retrieval and food sharing on the global activity of ant colonies as well to assess the ability of each colony to recover its initial activity baseline at satiation. The experiment was performed on a total of ten ant colonies.

Data collection
Video recordings were done with the iSpy1 v7.1.2.0. software and two Logitech C9201 webcams. A first webcam was placed 13cm above the nest ceiling and the second webcam was positioned 33cm above the foraging area (Fig 1). Videos were taken at 15 frames/sec at a 1920x1080 pixels resolution. On each video recording, the level of ants' activity was extracted using OpenCV (Open-Source Computer Vision v4.1.0) library in a Python1 (v3.7) script. On video recordings, any movement of the ants resulted in local changes in the brightness of the pixels. The script extracted the number of pixels that changed in brightness between each successive video frame and stored this value as a direct measure of ants' activity. To do so, the image was cropped to keep only the area of interest, here the nest area of 5.5x5.5cm, then was converted into a grey scale to keep only the black ants over a white background. A threshold was set to detect pixels that switched from black to white (and vice versa) and to record it as "activity". The activity measured in the foraging area used the same procedure of automatized image analysis except for the cropping that was focused on a 22x30.5cm area of the experimental tray. Furthermore, we verified that the level of global activity that was automatically detected did not simply reflect changes in the number of ants inside the nest but actual differences in workers' mobility. Thus, during the first phase of satiation, for each colony and each day at 9am, we counted the number of ants inside the nest and the number of foragers. The term "forager" is here referring to any ant observed outside the nest regardless of the task it performed on the foraging area. These numbers were compared to the activity levels measured on the corresponding video recordings made at 9am inside the nest and on the foraging area. Both inside and outside the nest, spearman correlation values were small and not statistically significant (S1 Table), indicating that, when food was given ad libitum, changes in the number of ants was not the main explanatory variable for differences in colony activity level.
Ants' mortality was recorded during the whole duration of the experiment. Nine out of ten colonies that were initially tested showed little mortality as well as a similar numbers of dead workers. The average mortality was 0.38 ± 0.64 (average dead ant ± standard deviation; n = 9) dead ant per day, which means that on average only 6.1 ± 2.7 (n = 9) ants had died per colony over 16 days (24h of habituation and 15 experimental days). For unexplained reasons, one colony however showed an unusually high level of mortality (38 total dead ants) and had to be taken out of the data analyses.

Data analyses
All statistical tests were done using the R software v3.3.1. and the significance threshold was set at α = 0.05.
Activity inside the nest and in the foraging area were recorded with two-minute-long videos, every 10 minutes. We obtained a total of 144 activity values per day (6 per hour for 24h) and per colony, creating the main database for further analyses of activity patterns.

Daily activity levels
To compare the average level of activity across colonies and over the course of the experiment, we calculated daily activity indices for each colony. Each daily index was the average of the 144 activity values measured on all two-minute videos that were recorded on this day. Moreover, we estimated to which extent the daily activity levels inside of the nest were linked to those observed in the foraging area by calculating the Spearman correlation value on each colony. This was done only for the starvation period since it was the only phase providing a sufficiently high number of successive daily observations.
We analyzed the activity patterns on each of the three phases of the experiment (Fig 2), the first phase of colony satiation when food was given ad libitum, the second phase of starvation when the colonies were food deprived and the third phase of recovery of satiation when food was given back to the colonies. On these daily activity indices, we visually checked on q-q plots the normality of the distribution of residuals. The homogeneity of variance of activity indices was checked by using a Bartlett test. All the distribution of daily indices were homoscedastic for each phase when looking at the activity in the inside and the outside of the nest. We used two-way ANOVAs to test the effect of phase, colony, and interaction of both factors on the daily activity indices. When we found a significant effect, we performed pairwise comparisons by using the Tukey HSD method. Within each phase, we also analyzed how the activity level of one colony changed across the successive days. Therefore, we performed a one-way repeated measures ANOVA to check for significant changes in activity levels over time. When significant, the test was followed by multiple paired t-tests between each day. P-values were then adjusted using the Bonferroni correction for multiple comparisons. These statistical analyses were performed for the activity levels measured inside the nest and also on the foraging area.

Synchronization of activity
We took a closer look at within-day fluctuations of activity in order to identify moments of synchronized activity/inactivity among nestmates. A high degree of synchronization means that a large proportion of individuals are active (or inactive) at the same time. To estimate the level of ants' synchronization, for each day and each colony, we measured to which extent the activity level obtained at each time point fluctuated around the corresponding average daily value. This was achieved by calculating the coefficient of variation (CV), that is the standard deviation divided by the mean value averaged over the 144 activity values measured on this day. High values of CV corresponded to high numbers of individuals being simultaneously active (or inactive). Previous studies have assessed the level of synchronized motion in other insect species by using a related metric called the index of dispersion (Temnothorax rugatulus [47], Schistocerca gregaria [51], Temnothorax allardycei [52]). Synchronization was analyzed only for ants inside the nest but not for ants located on the foraging area. Indeed, the very low local density of foragers makes encounters and mutual activation scarcely observed and hence a synchronization of activity unlikely to emerge on the foraging area.
High CV values may result from a few activity bouts of high amplitude but may also reflect frequent small fluctuations that take place all over the course of the experimental day and that are more pertaining to some "activity noise". Thus, we did not limit our analysis of synchronization to the CV metric, but we complemented it by a peak analysis in order to discriminate actual events of workers' synchronization. We used the R function findpeaks to automatically detect the activity peaks and to identify their time of emergence over each daily recording of activity. These functions detected peaks in each 24h-long time series depending on whether the measured activity level exceeded preset threshold values of prominence. Here, a peak was defined as a local maximal value within a set of three consecutive values, where this value had to be at least 10% higher than the average activity index for that day. Prior to the detection of local maxima, the time series was smoothed using a rolling average method with a rolling window of 3. We then counted the number of peaks detected on each day.
All the CV values inside the nest showed a normal distribution and were homoscedastic within each phase of the experiment. Likewise, the total number of peaks for each colony and for each day were normally distributed and homoscedastic within each phase of the experiment. Thus, we performed two-way ANOVAs to test the effect of phase, colony, and the interaction of both factors on the daily CV as well as on the number of activity peaks. When a significant effect was found, we performed pairwise comparisons by using the Tukey HSD method. Within each phase, we also analyzed how the CVs or the number of peaks changed across the successive days by performing a one-way ANOVA for repeated measures. If significant, we carried out multiple paired t-tests between each day whose P-values were adjusted using the Bonferroni correction method.

Periodicity of activity peaks
A high level of synchronization of ants' activity does not automatically go along with a periodicity of their activity pattern. For example, when a majority of nestmates are simultaneously moving during activity bursts, a colony can be seen as highly synchronized, even though these peaks do not occur at regularly spaced time intervals. In this respect, we carried out wavelet analyses on each daily activity values in order to evaluate the rhythmicity of oscillations of activity inside the nest. This technique, like a Fourier spectral decomposition, provides a breakdown of the dominant periods in the signal that we are studying. However, while a Fourier spectral decomposition transforms the whole signal into oscillatory components, the wavelet analysis fits a short oscillation that is tested at every point of the signal, thus giving it an extra spatial component. Once this oscillation is tested on the entire signal, the process is repeated with an oscillation of longer period and so on until all possible periods are tested. Wavelet analyses are therefore more robust when the signal is non-stationary, i.e., a signal whose properties change over time, for instance, if there is a trend in its mean value. As a result of this wavelet analysis, we obtain the continuous wavelet transform (CWT) that gives the spectral power associated with a certain period, at a certain position of the time series. High power values indicate a stronger contribution of the corresponding period to the oscillations shown by the activity signal. For instance, days where ant colonies showed a high rhythmicity of activity patterns are characterized by high maximum values of wavelet power. After decomposing the periodicity of the signal at each time step, we looked at which period was the most dominant for each colony and each phase. These periods followed a normal distribution and were homoscedastic within each phase of the experiment. Thus, we performed a two-way ANOVA to test the effect of phase and colony to see if the most dominant period was influenced by the satiation level and if it was colony dependent. As we were interested in ultradian rhythms, we only considered periods that did not exceed 24h. The wavelet analysis was carried out only on the activity patterns observed inside the nest but not in the outside as foragers were too scattered to show any periodic fluctuations of activity.

Daily activity levels
We found no significant interaction between colony and experimental phase on daily indices of activity inside the nest (ANOVA: Phase � Colony effect: F = 1.28, df = 16, P = 0.225). Interestingly, M. rubra colonies significantly differed in their activity level inside the nest throughout the experiment (ANOVA: Colony effect: F = 37.15, df = 8, P<0.0001). High levels of activity were consistently found in some colonies such as colony 2 that was always twice more active than colony 7 ( Fig 3A). We also found a significant effect of the experimental phase on the activity level inside the nest (ANOVA: Phase effect: F = 89.82, df = 2, P<0.0001). During the starvation phase, the daily activity indices were significantly lower (around 20%) than observed in satiated colonies (Tukey HSD: P1-P2 and P2-P3: P<0.0001; full comparisons with confidence intervals in S3 Table). During the recovery phase, just after food was given back to starved colonies, their activity indices increased and even exceeded by around 10% the activity initially during the first phase of colony satiation (Tukey HSD: P1-P3: P = 0.0017; S3 Table). When analyzing separately the first experimental phase, the activity indices of satiated colonies did not significantly change over time (ANOVA repeated measures: F = 0.52, df = 3, P = 0.674). Conversely, we observed a significant decrease of activity level over the successive days of starvation (ANOVA repeated measures: F = 47.15, df = 6, P<0.0001). Indeed, activity levels were significantly lower after only two days of starvation (S4 Table) with a 36% decrease over the whole period of food deprivation. Once food was given back, ant colonies first displayed the highest level of activity, which then gradually decreased with the recovery of colony satiation (ANOVA repeated measures: F = 9.08, df = 2, P = 0.00232) with indices significantly lower on day 15 compared to day 13 (S4 Table).
Outside the nest, the activity levels differed between colonies (ANOVA: Colony effect: F = 90.86, df = 8, P<0.0001). The activity of foragers also changed depending on the experimental phases (ANOVA: Phase effect: F = 27.45, df = 2, P<0.0001). However, unlike the inner workers, the activity of foragers did not differ between satiated and starved colonies (Tukey HSD: P1-P2: P = 0.372; S3 Table). The main change occurred during the last phase when colonies could again exploit food. Indeed, their activity indices became 28% and 36% higher than in the first and second phase, respectively (Tukey HSD: P1-P3 and P2-P3: P<0.0001; S3 Table). Furthermore, we found a significant interaction effect (ANOVA: Phase � Colony effect: F = 2.89, df = 16, P = 0.0007), meaning that certain colonies adjusted their external activity to food availability in a different way. Indeed, when being starved some colonies steeply increased their foraging activity in the outside (e.g., colony 4) and others showed only minimal variations (e.g., colonies 1 and 8). Also, during the last recovery phase, two colonies (5 and 7) did not increase their external activity as observed for the other nests (Fig 3B). After removing these two colonies from the analysis, there was no longer any significant interaction effect (ANOVA: Phase � Colony effect: F = 1.80, df = 12, P = 0.062). Looking separately in each experimental phase, we found a significant decrease of the external activity during the first phase of colony satiation (ANOVA repeated measures: F = 8.9, df = 3, P = 0.0004) with a lower activity of foragers on day 4 compared to day 1 (S5 Table). During the period of starvation the activity level of foragers slightly changed over time (ANOVA repeated measures: F = 2.48, df = 6, P = 0.0357) but no significant difference was found between any pair of starvation days (S5  Table). Concerning the recovery phase, the daily indices of activity outside the nest did not significantly change over time (ANOVA repeated measures: F = 0.086, df = 2, P = 0.918).
We found no clear-cut relationship between the daily activity indices that were measured inside and outside the nest of the same colony. These two activity values were not significantly correlated excepting for two colonies where a decrease of inner nest activity went along with a higher foraging during the starvation phase (S2 Table).

Synchronization of activity
For each colony, we examined how the activity values fluctuated across successive measures made every 10min over a 24h-period. Activity bouts of high amplitude reflected a high synchronization of workers that became active simultaneously. On each recording, the time series exhibited considerable variations of activity values from one hour to the next as well as across successive days. The activity patterns also varied between conditions of satiation and food shortage. During the initial phase of satiation, fluctuations of activity values remained of small amplitude and no clear-cut pattern of synchronized activity could be visually noticed (Fig 4A). Even a 24-hour periodicity of activity bouts could not be detected inside or outside the nest. Fig 4 provides an example of activity signals that are typically recorded during the first phase of the experiment, when food was given ad libitum. When colonies were starved, after two or three days of food deprivation, they exhibited quite different patterns of activity over a 24hperiod. Beside a decrease of their average activity level, starved colonies showed fluctuations of higher amplitude than those observed in satiated nests (Fig 4B).
When nestmates were highly synchronized, they showed several activity bouts of which the frequent occurrence resulted in high values of CV measured on the corresponding day. Interestingly, we found a significant Colony effect on daily CV suggesting that some nestmates were more likely to synchronize their activity inside the nest (ANOVA: Colony effect: F = 7.5, df = 8, P<0.0001). As illustrated in Fig 5, Table). Initially, satiated colonies showed moderate bursts of activity with an average CV of 22.6% ± 3.6% (average CV ± standard deviation; n = 36) that did not change over time (ANOVA repeated measures: P1: F = 0.45, df = 3, P = 0.719). When ant colonies were food deprived, these coefficients of variation became higher-even doubled for colony 7-reaching on average a 29.1% ± 8.8% (n = 63) value- (Fig 5). The amplitude of activity bursts, hence the level of synchronization, increased with colony starvation (ANOVA repeated measures: P2: F = 13.94, df = 6, P<0.0001). On the first day of starvation, day 5, the ants in the nest displayed a significantly lower CV than on day 9, 10 and 11 (S6 Table). On day 6, the CV values were significantly lower than on day 9 and 10. The activity became the least synchronized during the last phase. While sharing food inside the nest, nestmates showed only few activity bursts of small amplitude resulting in low CV values of 16.2% ± 3.6% (n = 27) that did not change over time (ANOVA repeated measures: P3: F = 3.23, df = 2, P = 0.0664). We also noticed an interaction effect between the colony and the phase (ANOVA: Phase � Colony effect: F = 2.48, df = 16, P = 0.0033). This interaction effect could be explained by the outlying fluctuations of activity in two colonies (3 and 7) during the starvation phase (Fig 5). Once these colonies were removed from the analysis, there was no longer any interaction effect (ANOVA: Phase � Colony effect: F = 1.01, df = 12, P = 0.445) while the colony and phase effects remained significant (ANOVA: Colony effect: F = 3.47, df = 6, P = 0.0044 | Phase effect: F = 34.17, df = 2, P<0.0001).

Occurrence of activity bursts
By using a peak analysis, we counted the activity bursts that occurred inside each colony on each experimental day (Fig 4C). Colonies did not differ in their number of daily activity peaks (ANOVA: Colony effect: F = 1.78, df = 8, P = 0.0901) and there was no interaction effect (ANOVA: Phase � Colony effect: F = 0.7, df = 16, P = 0.789). By contrast, the occurrence of activity peaks significantly changed between experimental phases (Fig 6) (ANOVA: Phase effect: F = 22.2, df = 2, P<0.0001). During the first phase of colony satiation, colonies showed on average 10 ± 2.3 (average number of daily peaks ± standard deviation; n = 36) activity peaks per day, with no significant change over time (ANOVA repeated measures: P1: F = 0.175, df = 3, P = 0.912). By contrast, during phase 2, starvation gradually increased the occurrence of activity bursts (Tukey HSD: P1-P2: P = 0.033; S3 Table). The maximum average daily values of 13.3 ± 1.5 (n = 9) and 11.9 ± 1.6 (n = 9) peaks were reached on days 10 and 11 respectively  Table). During the recovery phase, when colonies gained access to food again, the number of detected activity peaks became lower (Tukey HSD: P1-P3: P = 0.0005 | P2-P3: P<0.0001; S3 Table), reached at most 8 daily peaks on average and did not significantly change over time (ANOVA repeated measures P3: F = 3.19, df = 2, P = 0.0684).

Periodicity of activity patterns
Wavelet analyses of time series usually failed to show any activity cycles inside M. rubra nests. The wavelet analysis showed many peaks at different frequencies. There is no clear-cut periodicity in the daily patterns of activity. Indeed, out of the nine studied colonies and for the 15 days of experiment, so out of 144 time series, we identified only three occurrences of periodicity that lasted for around 10 hours. These three events of periodicity occurred in colony 1 on day 9, colony 2 on day 6 and colony 3 (Fig 7C) on day 3, which showed a period of around 160min, 180min and 170min, respectively. Moreover, the latter also displayed a periodicity of around 70min, from 11am to 3pm (Fig 7C). Activity cycles were thus scarcely observed and showed no consistency in terms of colony, phase or days involved. Even though there was noclear cut periodicity of activity patterns, a wavelet analysis performed on each phase allowed to extract the periods that had the overall highest power for this phase. For instance, for colony 7 during the starvation phase, three periods had overall higher power values (Fig 8). Thus, one can also extract, for each phase, the period that was associated to the highest power value (Fig  9). In each phase, the dominant periods markedly varied across colonies. For instance, during the first phase, the dominant period extracted from wavelet analyses ranged from 2h50 (colony 3) to 23h32 (colony 5). Likewise, the dominant period ranged from 1h52 (colony 6) to 22h48 (colony 7) during the second phase and from 5h04 (colony 9) to 23h14 (colony 2) during the last phase. Beside a weak and highly variable periodicity of activity patterns, we found no significant colony effect (ANOVA: Colony effect: F = 0.83, df = 8, P = 0.590) or effect of starvation on the dominant periods resulting from wavelet analyses (ANOVA: Phase effect: F = 0.48, df = 2, P = 0.625).

Discussion
We found that the average activity level inside M. rubra nests is a colony-specific trait. Between-colony differences in the activity level persisted over several days: colonies that were most active under the stable satiation condition remained so when being starved and when food was reintroduced. As the experimental colonies contained standardized numbers of queen, workers, and larvae, intercolonial differences in the activity level likely did not stem from differences in brood demand or tasks to be performed inside the nest. Instead, our results suggest that the activity level is specific to each ant colony and consistent across situationshere, along a gradient of food availability and colony starvation. In analogy to the individuallevel behavioral syndromes defined by Sih et al. [53,54], behavioral syndromes, including the carrying over of average activity across different contexts (e.g., in the presence/absence of resources, competitors, or predators), may occur at the colony level. In addition, persistent colony-and regional-level variation in foraging behaviors such as extra-nest activity, exploration, resource discovery and recruitment has been observed in harvester ants [15] and fire ants [35]. Differences in activity among colonies may arise from settlement in natural biotopes with different biotic and abiotic conditions. In the present study, the experimental colonies were maintained in the laboratory for at least 1 month before being tested, thereby reducing the impact of the ants' former living conditions. Colony-level variation in the activity level may also result from the past and current selection of heritable traits related to colony behavior. Variation in activity has been shown to reflect differences across colonies in gene expression in harvester ants [55] and fire ants [14]. Colony activity may also correlate with genetic diversity. For instance, harvester ant queens' engagement in multiple mating promotes genetic diversity in colonies that forage earlier in the day and for longer time periods [56]. In this respect, the ant species, M. rubra is characterized by variability in the degree of polygyny and number of patrilines, resulting in pronounced diversity of nestmate relatedness across colonies [48][49][50]. This diversity may, in turn, at least partially explain differences among colonies in the emergence of collective cooperative behavior and hence in the average colony-wide activity level. Comparative studies of ant colonies founded by lone single-mated queens and those founded by several multi-mated queens would enable the examination of whether a lesser degree of nestmate relatedness is accompanied by a lower average in-nest activity level, in the same way that Wiernasz et al. [56] examined the relationship of workers' relatedness to foraging efficiency.
In this study, starvation was not related to any systematic trend or change in foraging activity outside of the nest. In most colonies, the foraging activity remained unchanged even after a week of food deprivation. Only a subset of M. rubra colonies increased their foraging efforts under starvation stress, as reported previously for other ant species [20,29,57]. By contrast, all colonies gradually reduced in-nest activity when deprived of food resources. This energy-saving strategy may contribute to colony homeostasis by reducing the global energetic expenses inside the nest to meet lower nutrient incomes due to food shortage. Furthermore, the restriction of activity reduction to the inside of the nest preserves the ability of foragers to engage in timeframe (from 10am to 12pm) during which a period with a high spectral power (red on the right colored scale) was detected. During this narrow timeframe, the activity fluctuated with a periodicity of around 30min. (C) The graph of the CWT showed that the signal was periodic over a longer timeframe of around 8 hours (from 9am to 5pm). In this case, we found a dominant period of around 170min (2h50) that was complemented at 11am by a second shorter period of around 70 minutes.
https://doi.org/10.1371/journal.pone.0273087.g007 external activity to discover new resources. When the starved M. rubra colonies regained access to food, the activity levels inside the nest increased steeply, with ant movements likely linked to food sharing, which may involve up to 90% of the worker population as shown in other ant species [58,59].
A similar activity-reduction strategy has been observed in colonies of the ant species T. rugatulus under several months of food deprivation [46], in contrast to its application after only 1 week in the M. rubra colonies. This difference could be explained by differences in the species' metabolic rates and hunger levels: the slow pace of life of Temnothorax workers probably contributes to their incredible resistance to prolonged starvation, whereas the wakefulness of M. rubra workers is accompanied by a higher global metabolic rate, which may have led the colonies to quickly adopt the energy-saving strategy until the return of more favorable conditions. We assume that the lowering of activity as a response to food shortage is a strategy shared by many ant species but operating over very different time scales.
The colony specificity of the activity level demonstrated in this study supports the idea that ant societies act as coordinated, homeostatic systems characterized by specific levels of wakefulness and inactivity. Consistency in the average activity level does not preclude the occurrence of colony-wide fluctuations, including temporary activity bouts (regularly oscillating or not), over the course of the day. Under satiation, the M. rubra activity patterns included moments of synchronization, when many ants moved simultaneously. However, these activity peaks did not occur at regular intervals (range, 20 to 640 min). Starvation not only reduced the average activity level, but also increased the degree of activity synchronization. In colonies starved for 1 week, the number of activity bouts increased slightly while the synchronization metric (i.e., coefficient of variation) nearly doubled. This finding is consistent with the increased degree of synchronization and more frequent activity peaks reported for starved T. rugatulus colonies [46], although those colonies showed a greater increase in the number of activity peaks than observed in the present study, likely due to the longer starvation period. Differences in the peak detection technique may also have contributed to this dissimilarity. In the present study, the reintroduction of food to the starved colonies increased the average innest activity level and dramatically reduced the degree of nestmate synchronization, resulting in fewer activity bouts of lower amplitude. One possible explanation for these findings is that the signal noise caused by the erratic movements of workers actively sharing food inside the nest masked the detection of activity bursts.
The synchronization of activity found inside M. rubra nests is a common trait shared with other ants studied so far (Temnothorax allardycei [21], Leptothorax acervorum [44], and T. rugatulus [47]). The entrainment of nestmates that are roused (become motionless) following contact with active (inactive) workers creates positive feedback loops leading to the emergence of activity (inactivity) peaks. The role of synchronization as an epiphenomenon of mutual entrainment, however, does not prevent this characteristic from having functional value. Some authors point towards activity rhythms serve to regularly lower respiratory carbon-dioxide levels in nests [43]. Others evoke an impact on the colonies' ergonomic efficiency. By being active together may generate local overcrowding and queuing phenomena that reduce task performance efficiency [21] or, alternatively, prompt individuals to search for unperformed tasks, thereby favoring spatially more-homogenous task distribution across the colony [44,45]. A last hypothesis is that the synchronization of activity may facilitate the transmission of information or materials among nestmates [20]. However, not only information transmission, which is enhanced by synchronized activity peaks, but also information loss, which occurs when an ant forgets relevant information or when chemical information decays, should be considered. The interplay between these two mutually antagonistic feedback loops may lead to the reduction (to zero) or growth (to a steady state) of the population of informed ants. A data-driven model developed by Richardson et al. [19] predicts that information flows through a colony less efficiently when bouts of synchronized activity are periodic. This typically occurs when the lifetime of the information is shorter than the duration of quiescent activity periods, in which case information loss during quiescent periods dominates information spread during active periods. As we found that in-nest activity did not oscillate periodically in the majority of M. rubra colonies during starvation or satiation, the flow of information and materials among M. rubra nestmates is not expected to be impeded by regularly oscillating activity cycles. At the same time, synchronization of the activity of a sufficient number of M. rubra nestmates would enable the efficient completion of cooperative tasks and ensure reliable information exchange (as suggested by Franks and Bryant [60]). Under starvation stress demanding energy preservation, the greater synchronization of workers allows for long periods of inactivity while maximizing task performance and keeping communication efficient during temporary-but not periodic-bouts of activity.
The short (15-30 min) activity cycles of Temnothorax colonies serve as an example of selforganized periodicity that is not driven by an exogeneous pacemaker or the inherent rhythmicity of individual ants [21,61]. This remarkable self-organized behavior may have led to overestimation of the occurrence and importance of such activity cycles in insect societies. Few rhythms were observed inside M. rubra nest, and those that occurred were of longer duration (150-180 min) than reported previously. The very low occurrence rate for periodic ultradian patterns suggests that the rhythmicity of activity has little, if any, impact on M. rubra fitness. Similarly, several Temnothorax colonies [20,47] did not display periodic patterns, or displayed more erratic oscillation than others. Furthermore, the periodicity of activity cycles can be disrupted by several social factors, such as the return of foragers [22], location of interacting nestmates [22], high density of workers and brood [47], brood developmental stage [62,63], and ratio of castes within a colony [41]. Regularly spaced oscillations of activity resulting from coupled interactions between workers thus seem to emerge only under very specific conditions [21,64]. Among the many conditions required for the colony-wide propagation of an activation wave at regular time intervals are the appropriate density of mutually entraining ants [21], the homogenous spatial distribution of nestmates with no clear-cut aggregates [22], and a simple nest structure with no compartmentalization. As the natural nests of most ant species lack these traits, activity cycles appear to be striking periodic phenomena whose emergence is limited to artificial laboratory conditions. Thus, the rhythmicity of activity appears to be an exception, rather than the rule, in insect societies, and its adaptive value is likely marginal.
Our knowledge about the causes and functional value of activity patterns in ant colonies remains limited, considering the number of unresolved questions and the potential importance of these patterns for the behavioral ecology of social insects. Caution should be taken when attributing adaptive value to activity patterns observed under standardized laboratory conditions. In addition, the functional outcomes of activity levels are difficult to predict, and their impact on colony fitness may depend on the environmental conditions. On the one hand, colony-level variation in extra-nest activity has been found to correlate positively with markers of colony productivity, such as food storage and brood rearing, in honeybees [37] and with population growth in fire ants [35]. On the other hand, an elegant field study of Pogonomyrmex barbatus harvester ants showed that an increase in colony activity is not necessarily associated with improved fitness, as less-active and supposedly less-competitive colonies had the greatest reproductive success under harsh environmental conditions [15]. In addition, a better understanding of the effects of the activity level on colony-wide communication and recruitment dynamics, which shape the ability of ant colonies to adjust their responses to changing environmental conditions, is needed [34]. Finally, future studies will have to evaluate whether a synchronization of activity among nestmates actually facilitates the regulation of innest tasks, thereby allowing a colony to develop an energy-preservation strategy, namely under conditions of food shortages.
Supporting information S1 Table. Spearman correlation tests between the activity measured at 9am in one zone (nest or foraging area) and the number of ants in the corresponding zone at 9am. For each colony, tests are performed using all the daily activity indices measured during the satiation phase. P-values in bold are <0.05. (DOCX) S2 Table. Spearman correlation tests between the daily activity indices measured inside and outside of the nest for each colony during the satiation phase (n = 4 per colony) and the starvation phase (n = 6 per colony). Spearman tests were not performed for the recovery phase due to the too few daily activity indices available per colony. P-values in bold are <0.05.